Anne-Marie Koop

2 31 METHODS Animal model and study design Wistar rats (n=21; allmale; 160-180g; Charles River, the Netherlands) were randomized into 2 groups: sham (CON, n=7) or pulmonary artery banding (PAB, n=14, two of which died during surgery). PAB was performed through a left lateral thoracotomy as described before, 16 however, with a tighter constriction (1.1mm vs. 1.3mm before). Sham surgery was similar to the PAB surgery, with the exception of the actual banding of the pulmonary artery. From the moment of PAB/sham surgery onward, the animals were daily checked for signs of clinical RV failure (see 2.2 Definition of clinical RV failure ). 18 When a rat was identified as developing clinical RV failure, echocardiography and pressure-volume analysis was performed: these rats are the PAB+Clinical Failure (PAB+CF) group (n=5). Four rats with a PAB that did not show signs of clinical RVF, yet also had decreased distance run during voluntary treadmill exercise were analyzed and terminated at the same follow-up duration (PAB minus Clinical Failure: PAB- group, n=4). At 11 weeks the remaining rats (7 CON and 3 PAB) were terminated. See figure 1a for the experimental set-up. A detailed description of the methods is provided in the online- only Data Supplement. Definition of clinical RV failure Rats were examined daily for clinical signs of RV failure according to a predefined ABCDE-checklist, 6,18 which includes Appearance, Activity, Bodyweight, Circulation (peripheral), Cyanosis, Dyspnea/tachypnea and Edema, Effusions (see online-only Data Supplement). Clinical RV failure was defined as presence of at least: inactivity and ruffled fur and severe dyspnea and palpable ascites. The presence of ascites and pleural effusion was in all cases confirmed after termination. Pressure-volume measurements, echocardiography, exercise Functional analysis of the RV was performed before termination using invasive pressure-volume measurements in anesthetized (isoflurane/air mixture, 5% induction; 2-3% maintenance; analgesia with buprenorphine 0.01 mg/kg s.c.), ventilated rats following thoracotomy using a conductance catheter as described before 18 and in the online-only Data supplement. Stroke volume as measured by echocardiography (in mL) was used to calibrate the conductance-derived stroke volume (in arbitrary units) derived from the conductance signal. Steady-state pressure-volume loops were used for calculation of all parameters. End systolic and end diastolic elastance were determined using the single-beat method; vena cava